Project Summary Serratia spp are opportunistic pathogens that have been linked to nosocomial infections, keratitis, endocarditis and other human infections. There is a lack of molecular studies on Serratia during the infection process. Advancing research on microbial pathogenesis and infection have been greatly facilitated by the use of microbe-insect and microbe-nematode model systems. The overall goal of this AREA proposal is to identify the virulence factors of Serratia sp. SCBI, an insect pathogen and nematode symbiont. This proposal uses genomic (RNASeq) and both forward (gain-of-function) and reverse (loss-of-function) genetic approaches to address this issue. The proposed research will allow the identification of genes directly involved in pathogenesis and provide insight into their regulatory mechanisms. The Specific aims of this project are: 1a) Using a gain-of-function approach, a SCBI fosmid library will be screened to identify toxic clones; 1b) Genes for the clones identified from 1a will be identified and their role will be confirmed by generating knockouts of the genes either in the fosmid clones or in wild type cells; 1c) Using a loss-of-function approach, a transposon mutant library will be screened for loss of potential virulence traits; 1d) Genes will identified for the mutants selected in aim 1c and these mutants will be genetically complemented to show causality; and 2) Global gene expression by RNASeq will be determined for the wild type cells under different environmental conditions including under mutualistic or pathogenic states. Furthermore, the global expression pattern of regulatory mutants identified in Aim1 will determined and compared to the parental wild type. The impact on human health of these studies will be: 1) Provide evolutionary clues on the emergence of a pathogen, 2) Allow a deeper understanding of host-microbe interactions. In addition, this study will make a significant contribution toward the infrastructure of biomedical research and development. Both graduate and undergraduate students will play an active role in this research project, which fulfills one of the objectives of the AREA program.